Investigation Title	Transcription profiling time series of Sinorhizobium meliloti in response to an osmotic upshift elicited by salt or sucrose	
Comment[Submitted Name]	Sanjuan S. meliloti response to an osmotic upshift	
Experimental Design	compound_treatment_design	time_series_design	transcription profiling by array	
Experimental Design Term Source REF	mo	mo	EFO	
Comment[ArrayExpressReleaseDate]	2006-08-17	
Comment[AEMIAMESCORE]	5	
Comment[ArrayExpressAccession]	E-MEXP-785	
Comment[MAGETAB TimeStamp_Version]	2010-08-11 17:42:07 Last Changed Rev: 13058	
Experimental Factor Name	dose	time	compound	
Experimental Factor Type	dose	time	compound_treatment_design	
Experimental Factor Term Source REF	
Person Last Name	Pérez-Arnedo	Becker	Olivares	Soto	Domínguez-Ferreras	Sanjuán	
Person First Name	Rebeca	Anke	José	María	Ana	Juan	
Person Mid Initials				J	
Person Email					ana.dominguez@eez.csic.es	
Person Phone					0034 958 181600	
Person Fax					0034 958 129600	
Person Address					C/ Profesor Albareda nº1	
Person Affiliation	EEZ-CSIC	Bielefeld University	EEZ-CSIC	EEZ-CSIC	Microbiología del Suelo y Sistemas Simbióticos	EEZ-CSIC	
Person Roles					submitter	
Person Roles Term Source REF						
Quality Control Type	
Quality Control Term Source REF	
Replicate Type	
Replicate Term Source REF	
Normalization Type	
Normalization Term Source REF	
Date of Experiment	
Public Release Date	2006-08-17	
PubMed ID	16916894	
Publication DOI	16916894	
Publication Author List	Dominguez-Ferreras, Ana; Perez-Arnedo, Rebeca; Becker, Anke; Olivares, Jose; Soto, Maria J.; Sanjuan, Juan	
Publication Title	Transcriptome Profiling Reveals the Importance of Plasmid pSymB for Osmoadaptation of Sinorhizobium meliloti 	
Publication Status	journal_article	
Publication Status Term Source REF	
Experiment Description	Time course of the S. meliloti response to an osmotic upshift elicited by salt (300mM or 400mM) or sucrose (500mM or 700mM)	
Protocol Name	P-MEXP-28680	P-MEXP-28681	P-MEXP-28699	P-MEXP-28682	P-MEXP-28683	P-MEXP-28684	P-MEXP-28685	P-MEXP-28687	P-MEXP-28889	
Protocol Type	grow	nucleic_acid_extraction	pool	labeling	labeling	hybridization	hybridization	feature_extraction	bioassay_data_transformation	
Protocol Description	Cells were cultured in minimal media containing glutamate and mannitol as nitrogen and carbon sources, respectively, to exponential phase (A600 = 0.4) and then the appropriate amount of NaCl or sucrose was added to the culture in order to reach the desired concentration of these compounds. An equivalent amount of fresh minimal media was added to the control, unstressed cultures. Cells were then incubated (30ºC, 200 r.p.m.) and aliquots were collected after 15, 30, 60 or 240 minutes.	RNA was isolated after mechanical disruption of the cells (Rüberg et al., 2003) using the RNesay Mini Kit and RNase-free DNase Set (Qiagen). RNA was then purified and concentrated using Microcon-30 filters (Millipore)	Total RNA isolated from 3 biological replicates was pooled prior to concentration and purification with Microcon-30 filters (Millipore).	Total RNA was reverse-transcribed to yield aminoallyl-labeled first-strand cDNA using Superscript II reverse transcriptase and a dNTP stock solution including aa-dUTP.<br> After reverse-transcription, RNA was hydrolyzed and the sample was purified using the CyScribe GFX Purification Kit (Amersham Biosciences).<br> Coupling of fluorescent dyes to the aminoallyl-labeled first-strand cDNA was followed by quenching of the remaining dyes with hydroxylamine and clean-up of fluorescently labeled targets using CyScribe GFX Purification Kit (Amersham Biosciences)	Total RNA was reverse-transcribed to yield aminoallyl-labeled first-strand cDNA using Superscript II reverse transcriptase and a dNTP stock solution including aa-dUTP.<br> After reverse-transcription, RNA was hydrolyzed and the sample was purified using the CyScribe GFX Purification Kit (Amersham Biosciences).<br> Coupling of fluorescent dyes to the aminoallyl-labeled first-strand cDNA was followed by quenching of the remaining dyes with hydroxylamine and clean-up of fluorescently labeled targets using CyScribe GFX Purification Kit (Amersham Biosciences)	Processing of QMT epoxy microarrays carrying 70mer oligonucleotides prior to hybridization (Adapted by Anke Becker, Department of Genetics, University of Bielefeld): Slides were washed for 5 minutes at room temperature in Rinsing Solution 1 (250ml MilliQ water + 250µl of Triton X100; dissolve at 80ºC for 5 minutes and cool down to room temperature), for 2 minutes at room temperature in Rinsing Solution 2 (500ml MilliQ water + 50µl HCl 37%), this step was repeated, then once at room temperature for 10 minutes in Rinsing Solution 3 (225ml MilliQ water + 25ml KCl 1M), then 1 minute at room temperature in MilliQ water. Slides were incubated for 15 minutes at 50ºC in prewarmed blocking solution (150ml MilliQ water + 40µl HCl 37% + 50ml 4xQMT Blocking solution). Finally wash slides for 1 minute at room temperature in MilliQ water and spin immediately in a microplate centrifuge at 1200 rpm for 3 minutes to dry.<br> Hybridization protocol: Dissolve the completely dried combined Cy3/Cy5-labeled targets in 50µl of DIG Easy Hyb + 1 µl of sonicated salmon sperm DNA (5 µg/µl) and incubate at 65ºC for 5-10 minutes. Place the dried slide into the slide chamber, apply 50 µl of the denatured target as a drop to the center of the slide and place a cover slip (22x60mm) carefully on it. All manipulations are carried out on a 42ºC warmed surface. Place lid of the slide chamber on the chamber, tighten the screws and place it on the bottom of a 42ºC water bath. Hybridize not less than 14 and not more than 18 hours at 42ºC.<br> Remove the slide with the cover slip from the chamber and quickly dump it into 2xSSC, 0.2% (w/v) SDS washing buffer prewarmed to 42ºC, thereby washing off the cover slip. Place the slide in a black plastic box with 2xSSC, 0.2% (w/v) SDS washing buffer prewarmed to 42ºC and shake for 5 minutes. Transfer to 0.2xSSC, 0.1% (w/v) SDS and shake at room temperature (at most 24ºC) for 1 minute. Repeat. Transfer to 0.2xSSC and shake at room temperature (at most 24ºC) for 1 minute. Repeat. Transfer to 0.1xSSC at 18ºC and shake for 1 minute. Spin immediately in a microplate centrifuge at 1200 rpm for 3-5 minutes.<br> Place dried slides in the dark for at least 20 minutes before scanning.	Processing of QMT epoxy microarrays carrying 70mer oligonucleotides prior to hybridization (Adapted by Anke Becker, Department of Genetics, University of Bielefeld): Slides were washed for 5 minutes at room temperature in Rinsing Solution 1 (250ml MilliQ water + 250µl of Triton X100; dissolve at 80ºC for 5 minutes and cool down to room temperature), for 2 minutes at room temperature in Rinsing Solution 2 (500ml MilliQ water + 50µl HCl 37%), this step was repeated, then once at room temperature for 10 minutes in Rinsing Solution 3 (225ml MilliQ water + 25ml KCl 1M), then 1 minute at room temperature in MilliQ water. Slides were incubated for 15 minutes at 50ºC in prewarmed blocking solution (150ml MilliQ water + 40µl HCl 37% + 50ml 4xQMT Blocking solution). Finally wash slides for 1 minute at room temperature in MilliQ water and spin immediately in a microplate centrifuge at 1200 rpm for 3 minutes to dry.<br> Hybridization protocol: Dissolve the completely dried combined Cy3/Cy5-labeled targets in 250µl of DIG Easy Hyb + 3 µl of sonicated salmon sperm DNA (5 µg/µl) and incubate at 65ºC for 5-10 minutes. Place the dried slide into the chamber and inject the denatured target when prompted by the ASP software. It takes 10.5 to 16.5 hours (depending on the program) until the experiment continues.<br> Remove the slide from the ASP chamber and quickly place it in 2xSSC, 0.2% (w/v) SDS washing buffer prewarmed to 42ºC and shake for 1 minute after removal of the last slide. Transfer to 0.2xSSC, 0.1% (w/v) SDS and shake at room temperature (at most 24ºC) for 1 minute. Repeat. Transfer to 0.2xSSC and shake at room temperature (at most 24ºC) for 1 minute. Repeat. Transfer to 0.1xSSC at 18ºC and shake for 1 minute. Spin immediately in a microplate centrifuge at 1200 rpm for 3-5 minutes.<br> Place dried slides in the dark for at least 20 minutes before scanning.	Scanning was performed using the ScanArray software with a pixel size of 10µm to generate the corresponding images.<br> Mean signal and mean local background intensities were obtained for each spot of the microarray images using the ImaGene 5.5 software for spot detection, image segmentation and signal quantification (Biodiscovery Inc., Los Angeles, CA, USA).	Mean signal and mean local background intensities were obtained for each spot of the microarray images using the ImaGene 5.5 software for spot detection, image segmentation and signal quantification. Unreliable features were removed (bad or empty spots). The remaining spots were considered for further analysis. The logarithm to the bases 2 of the ratio of intensities was calculated for each spot using the formula Mi = log2 (Ri/Gi). Ri = I(ch1i)- Bg (ch1i) and Gi = I (ch2i)- Bg (ch2i), where I (ch1i) or I (ch2i) is the intensity of a spot in channel 1 or channel 2, respectively. In our case, positive log2 ratios corresponded to genes induced after the osmotic upshift and negative log2 ratios, to repressed genes. The mean intensity was calculated for each spot Ai = log2 (RiGi)to raise to the power of 0.5 (Dudoit et al. 2002). A normalization method based on local regression that account for intensity and spatial dependence in dye biases was applied. Within a print tip group normalization was performed according to Yang et al (2002). Genes significantly up- or down-regulated were identified by t-statistics (Dudoit et al., 2002). Normalization and t-statistics were carried out using the EMMA 1.1 microarray data analysis software developed at the Center for Genome Research at Bielefeld University (Dondrup et al., 2003). The p-value stated in the table is the result of the t-test performed on as much replicates as stated in the "replicates" column.	
Protocol Parameters	max temperature;min temperature;	Extracted product;Amplification;		Label used;Amplification;Amount of nucleic acid labeled;	Amount of nucleic acid labeled;Label used;Amplification;	Chamber type;time;Quantity of label target used;Volume;temperature;	time;Volume;Chamber type;Quantity of label target used;temperature;	
Protocol Hardware								ScanArray 4000 [PerkinElmer]	
Protocol Software								ScanArray Express [PerkinElmer]	
Protocol Contact	
Protocol Term Source REF								The MGED Ontology	The MGED Ontology	
SDRF File	E-MEXP-785.sdrf.txt	
Term Source Name	The MGED Ontology		ArrayExpress	nci_meta	mo	EFO	The MGED Ontology	
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php		http://www.ebi.ac.uk/arrayexpress	http://ncimeta.nci.nih.gov/indexMetaphrase.html	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/	http://mged.sourceforge.net/ontologies/MGEDontology.php	
Term Source Version